New & Noteworthy

If you’ve ever put together something from IKEA you know it can be a bear. So many parts need to connect up together perfectly to build that new bookcase—if you tried to do it all at once you’d go crazy.

Complicated tasks are way easier to do if they are broken up into smaller chunks. This is true whether you are building a bookcase or a biochemical pathway. Image via Wikimedia Commons

Luckily the good folks at IKEA try to make it a bit easier (and more tolerable) by splitting the task into smaller, more manageable pieces. You can concentrate on one part without having to worry about the rest. Once that part is done you can work on the next part and so on. In the end you assemble all the pieces together into your new bookcase.

This is the approach Galanie and coworkers took in their recent Science paper where they engineered our favorite yeast S. cerevisiae to make a couple of different opioids. And it is a good thing they broke this problem up, because it was a way bigger undertaking than anything IKEA might have thrown at them. Engineering this yeast strain was a genetic tour de force.

The authors coordinated 21 different genes from mammals, plants, bacteria and yeast to get the opiate precursor thebaine made. And the semisynthetic opiate hydrocodone took an extra two genes for a grand total of 23! Trying to do all of these at once might have been very frustrating. Thank goodness they split this Herculean task into six (or seven for hydrocodone) smaller modules.

The first step was to get yeast to make (S)-reticuline, a key intermediate on the way to useful opiates. This took 4 modules made up of 17 different genes: six from rat, six from plants, four from yeast and one from bacteria.

And of course just putting these into yeast all at once would almost certainly have made a whole lot of nothing. Each gene needed to be selected from the right beast and then optimized to work in the yeast strain. Sometimes this meant picking the right variant from the right plant, and sometimes it meant mutating a gene to make it behave better. This all would have been overwhelming if the task weren’t split into four easier sections.

Even with all of their optimization, this iteration only made about 20 μg/liter of (S)-reticuline. They needed yeast to crank out more of this intermediate, so they designed a fifth module.

As its name implies, this “bottleneck” module was designed to overcome bottlenecks in the first four modules. After it was added to the strain, the yeast managed to make 82 μg/liter. This was something they could work with!

Except now they were stuck. They needed (R)-reticuline instead of the S form, but no one knew how poppies managed this feat. The gene that did this job hadn’t yet been discovered.

So Galanie and coworkers rolled up their sleeves and dug through plant transcriptome databases to find the gene they were looking for. They found a likely candidate, synthesized the gene in order to produce the enzyme, tested whether it could transform the S form of reticuline into the R form in vitro, and found that it could.

They could now make the right intermediate, which meant they could make their final module. As its name implies, this “thebaine” module would finally allow them to make the opiate precursor thebaine in yeast. This module consisted of their recently discovered gene and three other plant genes.

They had finally made thebaine from simple sugars in yeast! Except it didn’t work very well at all. There seemed to be a bottleneck right after the (R)-reticuline stage. Back to the drawing board!

Given where the bottleneck was, the researchers guessed correctly that the culprit was the SalSyn enzyme which converted (R)-reticuline to salutaradine. A Western blot showed three distinct forms of this enzyme in yeast and only one form, the lowest molecular weight one, when it was expressed in tobacco. Clearly something was happening to inactivate this protein in yeast.

A close look at the protein suggested yeast was glycosylating positions that it shouldn’t, and site directed mutagenesis of these sites confirmed this. The glycosylation was causing the protein to be sorted incorrectly so that it couldn’t do its job.

Unfortunately just mutagenizing away the glycosylation sites wasn’t good enough, because this severely affected the enzyme’s ability to do its job. So the researchers created a chimeric protein with parts of another P450 enzyme they knew did great work in yeast. After optimizing its codons for yeast, this chimera performed beautifully.

Now, finally, they had a yeast strain that could make thebaine. Not a lot of it, only around 6.4 μg/liter—but amazing nonetheless.

A yeast strain would have to be millions of times better at making opioids before a Walter White character could turn it into a profitable criminal activity. But the authors advocate for starting an open dialog on synthetic biology issues now, while there’s still time to deliberate. Image by Hecziaa via deviantart.com

A final module was added that consisted of two plant enzymes that converted thebaine to the drug hydrocodone. This monster strain could crank out around 0.3 μg/liter of hydrocodone. Yes, that is as puny as it sounds; one dose of painkiller for an adult would contain 5 mg of hydrocodone.

To be competitive with poppies, they need a 100,000-fold improvement to around 5 mg/liter. In talking with Dr. Smolke, it sounds like this could happen within a couple of years. After scaling up for production, voila! An entirely new source of opiates for pain relief.

Of course the elephant in the room is a Breaking Bad-esque scene where a yeast biologist grabs ahold of an opiate-producing strain and supplies various cartels with illegal drugs. Our Walter White wannabe wouldn’t be able to use the current strain, as he would need thousands of liters of yeast to produce a single dose of Vicodin.

But this scenario will be a real concern in the next few years. Which is why the Smolke lab has crossed every t and dotted every i in setting up and creating this strain. They have made it as difficult as possible for the wrong people to get their hands on it.

This strain represents a stunning achievement in synthetic biology. Move over poppies, there’s a new opiate producer in town.

Imagine you were designing a factory to make a very special product. You’d study the process carefully, buy the right equipment, and bring in the right people.

To make a tricky product you need to have the right factory, workers and machinery. And if you’re making opiate drugs, then yeast makes a great factory! Image from Wikimedia Commons

So if one step made a lot of dust, while another step had to be dust-free – you’d be sure to separate them into different rooms of your factory. And you’d make sure that the instructions were written in a language that your experts could understand!

In a new paper in Nature Chemical Biology, Thodey and coworkers designed a factory in just this way to make some very important molecules: the opiate drugs that millions of people rely on every day to control pain. Because of this new factory, opium poppies won’t be needed for making these drugs (although they’ll still be very pretty!). The factory’s location: inside cells of our favorite yeast, S. cerevisiae.

The researchers first tried to coax the yeast to produce the natural opiates morphine and codeine. They recruited experts in the field (or, from the field), taking three opium poppy genes for enzymes in the opiate synthesis pathway: thebaine 6-O-demethylase (T6ODM), codeine O-demethylase (CODM), and codeinone reductase (COR).

Of course, simply transforming yeast with a plant gene doesn’t do much good. Yeast and poppies don’t speak the same language at the transcription level (and even their translation dialects are hard to understand). So the researchers put the poppy genes under the control of efficient yeast transcriptional regulatory sequences such as promoters and terminators, and optimized their codons for yeast.

Thodey and colleagues tweaked the system to try to steer it in the direction of the products they wanted. They fed the yeast additional monosodium glutamate and glutamine to increase intracellular levels of 2-oxoglutarate, which is required during catalysis by the T6ODM and CODM enzymes. They also varied the relative expression levels of the three poppy enzymes by varying the copy numbers of their genes in yeast.

Although these tweaks improved things, almost half the product was still the undesirable neomorphine. To address this, the researchers looked even more closely at the details of the pathway.

When morphine synthesis is going right, the neopinone made by T6ODM spontaneously rearranges to the codeinone that COR uses to continue along the pathway. But if COR grabs the neopinone before there is time for the rearrangement, the end result of the pathway is neomorphine, which does no one any good.

When you design a factory, it’s important that your assembly line doesn’t move too fast! In the yeast factory, when neopinone gets to the COR enzyme too quickly, the end result is not what you want – although maybe not this messy.

Going back to their blueprint, Thodey and colleagues decided to separate T6ODM and COR into different parts of the factory, to allow more time for this rearrangement. They added a tag to COR that would direct it to the endoplasmic reticulum membrane, while T6ODM stayed in the cytoplasm. Now it would take longer for neopinone to reach COR, giving it plenty of time to rearrange into codeinone. Sure enough, morphine production went way up.

This was great, but the researchers decided to take it a step further. Semisynthetic opioids such as hydrocodone, oxycodone, and hydromorphone are medically useful because they work better in some cases than the natural opiates. Currently, these are produced by chemical modification of the opiates produced by poppies. Could yeast do this job too? Of course!

Turning to different expert workers, Thodey and colleagues tried expressing the enzymes NADP+-dependent morphine dehydrogenase (morA) and NADH-dependent morphinone reductase (morB) from the bacterium Pseudomonas putida* along with the poppy enzymes. Again, the process needed a lot of tweaking, more than we can describe here. But the end result was a strain that produced both hydrocodone and oxycodone.

Putting together all their results, the researchers were able to construct three yeast strains, each like an assembly line tailored for different products. One assembly line is optimized for codeine and morphine, another for hydromorphone, and one for hydrocodone and oxycodone.

The next steps will be to scale up this process to industrial levels, and also to construct yeast strains that carry out the entire process starting from simple sugars, rather than needing to be fed the precursor thebaine. Substituting yeast cultures for opium poppy fields will have a huge global impact that goes far beyond pharmaceutical production.

It’s important to note that this factory could never have been constructed without knowing how to make its fundamental building blocks. Basic research in yeast molecular biology and genetics, which may seem arcane to some, was essential to provide the knowledge necessary to express and manipulate these foreign genes in yeast. Just another reason that we’re “high” on yeast research!

* Read more about Pseudomonas putida, a bacterial workhorse with an appetite for all kinds of weird substances.